A 2004 U.S. Department of Energy report entitled “Top value added chemicals from biomass” has identified twelve building block chemicals that can be produced from renewable feedstocks. The twelve sugar-based building blocks are 1,4-diacids (succinic, fumaric and maleic), 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, and xylitol/arabinitol.
Building block chemicals are molecules with multiple functional groups that possess the potential to be transformed into new families of useful molecules. These twelve building blocks can be subsequently converted to a number of high-value bio-based chemicals or materials.
Many natural metabolites derived from biological fermentative processes such as dicarboxylic acids, amino acids, and diols have functional groups that are suitable for polymerization and chemical synthesis of polymers. In the recent years, the efficiency of microorganisms for producing monomeric chemical compounds suitable for industrial usage has been significantly increased through genetic manipulations. However, the cost of producing industrial chemicals through biological fermentative process is still very high. At present the biological fermentative processes for the production of industrial chemicals use purified carbohydrates such as glucose and corn starch as the source of carbon and thereby add cost to the fermentative process for producing industrial chemicals.
The cost of the fermentation process for producing industrial chemicals can be dramatically reduced by using lignocellulosic biomass as the source of carbon in the fermentation process. Lignocellulosic biomass can be obtained from a number of sources including agricultural residues, food processing wastes, wood, and wastes from the paper and pulp industry. Biomass consists of roughly 40-50% of hexose sugars (sugars with six carbon atoms) and 10-30% of pentose sugars (sugars with five carbon atoms). The hexose sugars are known in the art as C6 sugars. The pentose sugars are known in the art as C5 sugars. When hydrolyzed, the lignocellulosic materials yield a mixture of sugars that includes glucose, xylose, arabinose, mannose and galactose. However, a number of fermentation processes for the production of industrial chemicals have been developed with pure glucose as a source of carbon for their growth and metabolism. For example, the E. coli strain described in U.S. Pat. No. 7,223,567 uses a rich medium supplemented with glucose as the source of carbon. The E. coli strain KJ122 useful for the production of succinic acid described by Jantama et al (2008a; 2008b) and in the published PCT Patent Applications Nos. WO/2008/021141A2 and WO2010/115067A2 can grow on a minimal medium but still requires glucose or another sugar as the source of carbon. It would be ideal if these organisms with the ability to produce industrial chemicals at high efficiency could be grown in a mixture of sugars derived from hydrolysis of lignocellulose. The inventors have discovered a method to enable the microorganisms already optimized to produce a specialty industrial chemical to use a mixture of C5 and C6 sugars derived from hydrolysis of lignocellulosic feedstock.
The ability of the microorganism to use multiple sugars simultaneously is limited by the existence of certain biochemical regulatory systems. These biochemical regulatory systems within the microbial cells have a genetic basis. Efforts have been made to overcome these regulatory systems through genetic manipulations.
In many cases industrial microorganisms are grown in a medium containing glucose or sucrose as the source of carbon. The presence of glucose in the growth medium suppresses the use of other sugars in E. coli and other species of industrial microorganisms. The consumption of other sugars such as xylose, a pentose sugar, by these microorganisms is initiated only after glucose in the growth medium has been fully consumed. This phenomenon related to carbon utilization in industrial microorganisms is referred to as catabolite repression or diauxic growth. A method to make the microorganisms co-utilize the different sugars such as C5 and C6 sugars through a relief of catabolite repression during the production of industrial chemicals in a commercial scale would be critical to lowering the cost of industrial chemicals produced by fermentation.
Microorganisms take up sugars through a set of transporter proteins located in the cytoplasmic membrane. The microbial sugar transporters fall within three major categories. The largest group of sugar transporters in bacteria is known as ATP binding cassette (ABC) transporters. As the name implies, the ABC transporters require a molecule of ATP for every molecule of sugar transported into the bacterial cell. XylFGH is an ABC transporter for the transport of xylose, a pentose sugar, into the cell. AraFGH is an ABC transporter for the transport of arabinose, yet another pentose sugar.
The second type of bacterial sugar transporters are grouped under Major Facilitator Super family (MFS). Within the MFS sugar transporters, two different categories of transporter are recognized. MFS includes H+-linked symporters, Na+-linked symporters-antiporters and uniporters. The uniporters are simple facilitators for the sugar transport and do not require a molecule of ATP for every molecule of sugar transported into the cell. The trans-membrane protein Glf in Zymononas mobilis is an example of a facilitator. The H+-symporters require an extracellular proton for every sugar molecule transported into the cell. The GalP protein in E. coli is a symporter for the transport of galactose, a hexose sugar, into the cell. GalP is a very well characterized symporter with 12 trans-membrane loops. GalP is also reported to have the ability to transport glucose across the cell membrane. AraE is a proton-linked symporter for the transport of arabinose across the cell membrane. Similarly XylE protein is a proton-linked symporter for the transport of xylose.
The third sugar transporter primarily responsible for the uptake of hexose sugars such as glucose is known as the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). As a way of differentiating the other two sugar transport systems from PTS, the other two sugar transport systems (ABC transporters and members of MFS transporters) are referred as non-PTS sugar transporters. Transfer of the phosphoryl group from phosphoenolpyruvate (PEP) catalyzed by the PTS drives the transport and phosphorylation of glucose and other sugars and results in the formation of phosphorylated sugars and pyruvic acid inside the cell. PTS generated pyruvic acid is apparently not recycled to PEP under aerobic culture conditions where glucose is the sole source of carbon. Rather, pyruvate is oxidized by way of the tricarboxylic acid cycle to carbon dioxide. Thus, for the transport of every single molecule of glucose, a molecule of PEP is consumed. In terms of cellular bioenergetics, the transport of sugars through PTS is an energy intensive process. Therefore in cells growing anaerobically, where there is a need to conserve the phosphoenolpyruvate content within the cells for the production of industrially useful chemicals, it is desirable to replace the PTS with other non-PTS sugar transporters not requiring a molecule of PEP for every molecule of sugar transported into the cell.
The PTS is comprised of two cytoplasmic components named EI and HPr and a membrane-bound component EII. E. coli contains at least 15 different EII complexes. Each EII component is specific to a sugar type to be transported and contains two hydrophobic integral membrane domains (C and D) and two hydrophilic domains (A and B). These four domains together are responsible for the transport and phosphorylation of the sugar molecules. EI protein transfers the phosphate group from PEP to HPr protein. EII protein transfers the phosphate group from phosphorylated HPr protein to the sugar molecule.
EI is encoded by the ptsI gene. HPr is encoded by the ptsH gene. The glucose-specific EII complex of enteric bacteria consists of two distinct proteins namely, EIIAGlc encoded by the gene crr and the membrane-associated protein EIICBGlc encoded by the gene ptsG. The PTS mediated sugar transport can be inhibited by means of mutating one of these genes coding for the proteins associated with PTS. Functional replacement of PTS by alternative phosphoenolpyruvate-independent uptake and phosphorylation activities (non-PTS) has resulted in significant improvements in product yield from glucose and productivity for several classes of metabolites.
With the decrease in the PTS-mediated glucose uptake, other systems for glucose uptake can be activated to assure the continued availability of glucose within the cell for the production of the industrially useful chemicals. For example, the glf gene coding for glucose permease, a glucose uniporter, has been shown to substitute for the loss of PTS mediated glucose uptake. Similarly the over expression of galP and glk genes are reported to enhance the glucose uptake and phosphorylation in the pts− strain of E. coli. GalP is a symporter for the uptake of galactose, a hexose sugar. GalP has been reported to transport glucose in the pts− strain. The significance of GalP mediated glucose uptake is evidenced by the fact that the inactivation of galP gene in the pts− mutant is found to prevent growth on glucose (Yi et al., 2003). In the absence of a PTS, Glk is necessary to achieve the phosphorylation of the glucose molecule before it can enter into glycolysis. The expression of the GalP protein in a pts− stain can be achieved either by expressing the galP gene under a constitutive promoter or by means of relieving the repression of the galP gene expression through mutations in genes coding for the repressor of the galP gene such as galS and galR.
Besides reducing the energy cost incurred in the transport of sugars into the cells, the introduction of a mutations into a gene coding for a protein associated with PTS is expected to relieve the catabolite repression which in turn would allow the simultaneous transport and utilization of all the sugars present in the culture medium including the pentose and hexose sugars. Hernandez-Montalvo et al (2001) studied the utilization of a sugar mixture comprising glucose, arabinose and xylose by an E. coli strain devoid of PTS for the transport of glucose. The pts− mutant was able to uptake sugars by a non-PTS mechanism as rapidly as its wild-type parental strain. In cultures grown in minimal medium supplemented with glucose-xylose or glucose-arabinose mixtures, glucose repressed arabinose or xylose-utilization in the wild type strain. Under the same culture conditions, the pts− mutant co-metabolized glucose and arabinose. However, glucose still exerted a partial repressive effect on xylose consumption. In cultures growing with a triple mixture of glucose-arabinose-xylose, the wild type strain sequentially utilized glucose, arabinose and finally xylose. In contrast, the pts− strain co-metabolized glucose and arabinose, whereas xylose was utilized after glucose-arabinose depletion. As a result of glucose-arabinose co-metabolism, the pts− strain consumed the total amount of sugars contained in the culture medium 16% faster than the wild type strain.
A pts− mutant strain with the capacity to co-metabolize glucose and xylose would cause further increase in the rate of consumption of sugar in the medium leading to an increase in productivity. Thus there is a need in the art for a microorganism that could co-metabolize glucose and xylose since these two sugars represent the predominant sugars that are present in the raw cellulosic hydrolysate. Moreover, it has been reported that the elimination of the ptsG gene function could decrease the rate of growth of microorganisms metabolically engineered to produce organic acids (Sanchez et al., 2005). Therefore there is an additional need to achieve the ability to use multiple sugars simultaneously without compromising the growth rate and rate of production of commercially important chemicals and chemical intermediates.
The objective of the present invention was to metabolically evolve microorganisms capable of producing high levels of industrial chemicals using multiple sugars simultaneously without reducing the productivity. The inventors have surprisingly identified a process for making microorganisms that simultaneously consume multiple sugars through metabolic evolution. This process of metabolic evolution allows the cells to acquire the ability to use multiple sugars without affecting any of its original characteristics such as rapid growth, and the ability to produce specific industrial chemicals at commercially significant quantities.
The inventors have also identified a novel genetic basis for the ability of the microorganism to use glucose and xylose simultaneously. Whole-genome sequencing was used to identify the genetic modification that confers to the microorganisms the ability to use multiple sugars simultaneously in the production of organic acid.
Prior to the present invention, it would have been doubtful whether the ability to utilize both hexose sugars and pentose sugars simultaneously could be accomplished through a simple genetic manipulation. The present invention related to molecular genetics offers the potential to achieve the ability to metabolize efficiently the entire range of biomass-derived sugars. For the first time, the present invention provides a genetic approach for achieving simultaneous glucose and xylose uptake that is not obligately coupled to the expenditure of phosphoenol pyruvate.